This proposal is designed to elucidate molecular mechanisms of pathogenesis in Pseudomonas aeruginosa strains infecting patients with Cystic Fibrosis (CF). Following the initial infection, P. aeruginosa undergoes mucoid conversion and acquires the ability to cause chronic, debilitating, and life-threatening disease. The mucoid phenotype is due to the overproduction of a capsule-like exopolysaccharide called alginate, which is associated with resistance to phagocytosis and new adherence properties. Adaptive mutations occur in vivo to derepress algT-encoded alternative sigma factor-22, which also controls a global stress response. This leads to the activation of a regulatory cascade that includes the overproduction of a1ginate. This proposal addresses the elucidation of this bacterial stress response during pulmonary infection in CF. We will define the mechanism of sigma-22 posttranscriptional regulation by MucABCD, which apparently forms a membrane complex that controls sigma-22 turnover by an unknown signal transduction mechanism. Sigma-22 controls the genes of a stress response regulon that is probably activated upon infection, and it is hyperactivated upon mucoid conversion. We will define the genes under sigma-22 control that are activated upon mucoid conversion to better understand the response system that is so highly expressed upon mucoid conversion. The sensors (or receiver modules) called KinB and FimS are probably responsible for detecting important environmental conditions during infection, and then transmit this information to their regulators, AlgB and AlgR, respectively. This in turn activates the expression of genes that are probably involved in in vivo survival. To better understand these sensor-regulator systems, we will define the roles of these sensors of the 2-component systems under sigma- 22 control by elucidating the genes under their control. In addition, we have found that the rpoS encoded sigma factor sigma-S (or RpoS) controls alginate production, and so probably controls a step in the sigma-22 regulatory cascade. We will characterize the role of sigma-S in the alginate regulon and other genes it controls that are associated with the stress response during infection. The information gained from the analysis of this complex regulon, which is activated upon infection of the CF lung by P. aeruginosa, improve our understanding of this unique host-pathogen interaction and may lead to the development of successful therapy.